RPE-Directed Gene Therapy Improves Mitochondrial Function in Murine Dry AMD Models

Age-related macular degeneration (AMD) is the most common cause of blindness in the aged population. However, to date there is no effective treatment for the dry form of the disease, representing 85–90% of cases. AMD is an immensely complex disease which affects, amongst others, both retinal pigment epithelium (RPE) and photoreceptor cells and leads to the progressive loss of central vision. Mitochondrial dysfunction in both RPE and photoreceptor cells is emerging as a key player in the disease. There are indications that during disease progression, the RPE is first impaired and RPE dysfunction in turn leads to subsequent photoreceptor cell degeneration; however, the exact sequence of events has not as yet been fully determined. We recently showed that AAV delivery of an optimised NADH-ubiquinone oxidoreductase (NDI1) gene, a nuclear-encoded complex 1 equivalent from S. cerevisiae, expressed from a general promoter, provided robust benefit in a variety of murine and cellular models of dry AMD; this was the first study employing a gene therapy to directly boost mitochondrial function, providing functional benefit in vivo. However, use of a restricted RPE-specific promoter to drive expression of the gene therapy enables exploration of the optimal target retinal cell type for dry AMD therapies. Furthermore, such restricted transgene expression could reduce potential off-target effects, possibly improving the safety profile of the therapy. Therefore, in the current study, we interrogate whether expression of the gene therapy from the RPE-specific promoter, Vitelliform macular dystrophy 2 (VMD2), might be sufficient to rescue dry AMD models.


Introduction
Age-related macular degeneration (AMD) is a devastating and progressive degenerative disorder of the macula leading to loss of central vision. It is the leading cause of blindness in the elderly in the developed world, affecting~10% of people over 65 years of age [1]. AMD is a multifactorial condition with genetic and environmental factors known to contribute to the disease, with age being the greatest risk factor. Twin studies estimate the heritability of AMD at between 46-71% [2]. AMD is generally divisible into 2 distinct forms: neovascular (wet) and non-exudative (dry), accounting for 10-15% and 85-90% of cases, respectively [3].
In early dry AMD, drusen are formed between the Bruch's membrane (BM) and the basal lamina of the retinal pigment epithelium (RPE). In the late stage, the condition may progress to geographic atrophy (GA), characterised by atrophy of the photoreceptors, RPE and choriocapillaris in the macula. GA typically initiates in the perifoveal macula, but models with a gene therapy, ophNdi1 [38]. OphNdi1 is a modified gene from S. cerevisiae, NDI1, which has been optimised to express more efficiently in mammalian cells (patent no. 10220102). Encoded by a single nuclear gene, NDI1 performs a similar function to mammalian mitochondrial complex 1. OphNdi1, delivered subretinally via recombinant adeno-associated virus 2/8 (AAV2/8), was shown to provide significant and robust benefit in the Cfh −/− and NaIO 3 -induced murine models and two cellular models of dry AMD [38]. The study was the first to show functional benefit in vivo in murine models of dry AMD, using a gene therapy which directly targets mitochondrial dysfunction. In the study, AAV2/8-ophNdi1 was driven from generic CMV and CAG promoters so that ophNdi1 was expressed in both RPE and photoreceptor cells. In contrast, in the current study, we evaluated whether targeted treatment of the RPE with ophNdi1 was sufficient to provide benefit to dry AMD models, since it is thought that AMD may initiate in the RPE prior to photoreceptor cells [52]. In addition, restricting ophNdi1 expression to the RPE may reduce potential off-target effects in other cell types; thereby, in principle, improving the potential safety profile of the therapy. Hence, in this study we replaced the generic promoter (CMV or CAG) with a vitelliform macular dystrophy (VMD2) promoter, which, in the context of the eye, is known to restrict gene expression to the RPE [53], creating VMD2-ophNdi1. VMD2-ophNdi1 was expressed from recombinant AAV2/8 (AAV2/8-VMD2-ophNdi1; Figure 1) and its therapeutic potential was investigated in the Cfh −/− genetic and NaIO 3induced murine models of AMD. Primary porcine RPE (pRPE) models of dry AMD were also utilised; these were either insulted with NaIO 3 [38] or loaded with retinylidene-Nretinylethanolamine (A2E) and insulted with blue light [47]. A2E is a toxic bisretinoid byproduct of the visual cycle and a major component of drusen, which is known to lead to RPE dysfunction and cell death in vitro and in vivo [47,[54][55][56]. , which were utilised to generate AAV2/8-VMD2-EGFP and AAV2/8-VMD2-ophNdi1 viral preparations. The VMD2 promoter and EGFP and ophNdi1 transgenes, the minimal PolyA, ITRs and sizes are indicated. (C-E) Comparison of cell type specificity and EGFP expression level from the VMD2 and CMV promoters in murine retina. Two-month-old 129 S2/SvHsd mice were subretinally injected with 3 × 10 9 vg of AAV2/8-CMV-EGFP (C) or AAV2/8-VMD2-EGFP (D,E). Four weeks post-injection, eyes were enucleated, fixed in 4% PFA, cryosectioned, and processed for and analysed by fluorescent microscopy. Green and blue represent EGFP and DAPI (nuclear counterstain) fluorescence, respectively. Panels (C,D) were displayed using the full intensity range of EGFP fluorescence. Panel (E) corresponds to the same microscopy image as panel (D); however, in panel (E), the displayed EGFP fluorescence intensity range was focused on the lower intensity values, which enabled the visualisation of lower EGFP levels. EGFP fluorescence intensity levels were measured in the RPE for both promoters, and levels were found to be ~6.5-fold lower from VMD2 compared to CMV. RPE: retinal pigment epithelium, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bar for retinal sections (E) = 100 μm.

Rescue in pRPE Cell Models
To determine whether the VMD2 promoter-driven ophNdi1 therapy could rescue cell models of dry AMD, insulted primary pRPE cells were utilised. Primary pRPE cells isolated from n = 3 adult pigs were transduced with AAV2/8-VMD2-ophNdi1 (MOI = 3.4 × 10 6 ), insulted with 6 mM NaIO3 and compared to control cells. Cells were fixed 24 h post- Figure 1. Diagrammatic representation of the plasmid constructs VMD2-ophNdi1 (A) and VMD2-EGFP (B), which were utilised to generate AAV2/8-VMD2-EGFP and AAV2/8-VMD2-ophNdi1 viral preparations. The VMD2 promoter and EGFP and ophNdi1 transgenes, the minimal PolyA, ITRs and sizes are indicated. (C-E) Comparison of cell type specificity and EGFP expression level from the VMD2 and CMV promoters in murine retina. Two-month-old 129 S2/SvHsd mice were subretinally injected with 3 × 10 9 vg of AAV2/8-CMV-EGFP (C) or AAV2/8-VMD2-EGFP (D,E). Four weeks post-injection, eyes were enucleated, fixed in 4% PFA, cryosectioned, and processed for and analysed by fluorescent microscopy. Green and blue represent EGFP and DAPI (nuclear counterstain) fluorescence, respectively. Panels (C,D) were displayed using the full intensity range of EGFP fluorescence. Panel (E) corresponds to the same microscopy image as panel (D); however, in panel (E), the displayed EGFP fluorescence intensity range was focused on the lower intensity values, which enabled the visualisation of lower EGFP levels. EGFP fluorescence intensity levels were measured in the RPE for both promoters, and levels were found to be~6.5-fold lower from VMD2 compared to CMV. RPE: retinal pigment epithelium, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bar for retinal sections (E) = 100 µm.

Results
In order to estimate the dose of AAV2/8-VMD2-ophNdi1 ( Figure 1A) that might be effective in murine RPE, we generated an AAV2/8 vector expressing VMD2 promoterdriven enhanced green fluorescent protein (EGFP) gene (AAV2/8-VMD2-EGFP; Figure 1B) which could be compared in vivo to a similar CMV-driven EGFP vector (AAV2/8-CMV-EGFP). Two-month-old 129 S2/SvHsd mice were subretinally injected with 3 × 10 9 vg of either AAV2/8-VMD2-EGFP or AAV2/8-CMV-EGFP. Relative EGFP protein expressions from the two promoters were determined in mouse retinas four weeks post-injection. EGFP expression from the VMD2 promoter was restricted to the RPE, whereas the CMV promoter efficiently drove EGFP expression in the RPE and the outer nuclear layer containing the photoreceptors ( Figure 1C-E). Levels of EGFP fluorescence in the RPE were evaluated using fluorescent microscopy and were estimated to be 6.5-fold higher from the CMV than from the VMD2 promoter. Therefore, to account for this differential in expression, doses of AAV2/8-VMD2-ophNdi1 used in the current study, in both in vitro and in vivo models, were 6.5-fold higher than doses of the non-specific CMV promoter [38].
Two-month-old 129 S2/SvHsd mice were subretinally injected with 3 × 10 9 vg of AAV2/8-CMV-EGFP and AAV2/8-VMD2-EGFP ( Figure 1A). Four weeks post-injection, eyes were enucleated, fixed in 4% PFA, cryosectioned, and processed for and analysed by fluorescent microscopy. Green and blue represent EGFP and DAPI (nuclear counterstain) fluorescence, respectively. Panels C and D were displayed using the full intensity range of EGFP fluorescence. Panel E corresponds to the same microscopy image as panel D; however, in panel E, the displayed EGFP fluorescence intensity range was focused on the lower intensity values, which enabled the visualisation of lower EGFP levels. EGFP fluorescence intensity levels were evaluated in the RPE for both promoters and levels were found to be~6.5-fold lower from VMD2 compared to CMV.

Rescue in pRPE Cell Models
To determine whether the VMD2 promoter-driven ophNdi1 therapy could rescue cell models of dry AMD, insulted primary pRPE cells were utilised. Primary pRPE cells isolated from n = 3 adult pigs were transduced with AAV2/8-VMD2-ophNdi1 (MOI = 3.4 × 10 6 ), insulted with 6 mM NaIO 3 and compared to control cells. Cells were fixed 24 h postinsult and analysed with immunocytochemistry for 8-OHdG (oxidative stress marker), CPN60 (mitochondrial marker), phalloidin (selective for F-actin) and Hoechst (nuclear stain). NaIO 3 -treated cells exhibited high levels of oxidative stress and absence of actin filaments compared to non-insulted cells, indicating severe stress and reduced viability. In contrast, insulted cells transduced with AAV2/8-VMD2-ophNdi1 appeared more similar to non-insulted controls. Furthermore, mitochondrial staining was more intense and punctuated in insulted cells that had not received therapy, likely indicating mitochondrial dysfunction. Mitochondria of AAV2/8-VMD2-ophNdi1-treated cells insulted with NaIO 3 were more similar to non-insulted control cells, although staining was still somewhat elevated ( Figure 2A-O).

Discussion
We have previously shown that subretinally injected AAV-delivered ophNdi1 provided robust functional benefit and increased mitochondrial function in the Cfh −/− and NaIO 3 -induced mouse models of dry AMD. Additionally, increased cellular bioenergetics and reduced cellular stress markers were found [38]. In the prior study, ophNdi1 expressed from a recombinant AAV2/8 vector was driven from a ubiquitous CMV or CAG promoter and therefore was expressed in multiple retinal cells, including RPE, rod and cone photoreceptor cells; the cells lost in advanced dry AMD. In the current study, a higher absolute dose of AAV2/8-VMD2-ophNdi1 (4.5 × 10 8 vg) was utilised to match the effective dose range of AAV2/8-CMV-ophNdi1 (1 × 10 7 vg and 7.5 × 10 7 vg) and achieve comparable levels of expression of ophNdi1 from the RPE-specific VMD2 promoter, which was estimated to express~6.5-fold less than the CMV promoter in RPE cells ( Figure 1C-E).
There is strong evidence that AMD may initiate in the RPE, causing subsequent dysfunction in photoreceptor cells and ultimately, in GA, cell death in RPE and photoreceptor cells. However, this order of events is by no means definitively established. Differentially expressed genes have been identified in both RPE and cells of the neural retina and in both peripheral and macular regions of post-mortem AMD patient eyes [57]. In addition, rod photoreceptor cells also show early functional and histological signs of degeneration, as early signs of AMD include parafoveal scotomas and scotopic sensitivity; the parafoveal area is dense in rod photoreceptor cells [58]. Physiological abnormalities in cones in early dry AMD have also been widely reported and are indicative markers of the severity of dry AMD [59]. The morphological changes in cones in early dry AMD are, however, more subtle and include abnormal immunoreactivity to cone opsin, in combination with swelling of and altered immunoreactivity in the cone distal axon [60]. Thus, based on these features, many researchers believe that photoreceptor cell dysfunction may possibly occur in parallel with, or even prior to, dysfunction in the RPE/Bruch's membrane complex [61]. Hence, the exact sequence of disease progression remains somewhat obscure.
Experimental therapies for AMD have focused on preservation of photoreceptor cells, RPE or both [62][63][64][65][66][67]. However, clearly, in terms of optimising efficacy and safety and reducing possible off-target effects, there may be a therapeutic advantage to restricting expression of a gene therapy to cell types underlying the condition, with the aim of preventing the disease from progressing to other retinal cells. The aim of the current study was to determine whether expression of ophNdi1 solely in RPE is sufficient to rescue a variety of AMD cell and murine models. Primary RPE cells were insulted with either NaIO 3 or A2E/blue light. AAV2/8-VMD2-ophNdi1 was shown to rescue these cellular models using assays for ROS, cell viability, mitochondrial morphology and mitochondrial function (OXPHOS), in a similar fashion to CAG-driven ophNdi1 (Figure 2), indicating that the VMD2 promoter is functional and efficient in primary RPE cells.
In the Cfh −/− mice, ERG readings were significantly improved, and ROS levels reduced by, AAV2/8-VMD2-ophNdi1 treatment, as had also been seen in a previous study utilising AAV2/8-CAG-ophNdi1 in this model [38]. However, in contrast to AAV2/8-CAG-ophNdi1, AAV2/8-VMD2-ophNdi1 did not rescue cone numbers in the model, whereas the ONL showed a trend towards being thicker in treated eyes ( Figure 3). Additionally, in the very acute and severe NaIO 3 -induced murine model of dry AMD, only OKR benefit could be demonstrated with AAV2/8-VMD2-ophNdi1 (Figure 4), whereas AAV2/8-CAG-ophNdi1 had previously provided OKR, ERG and histological benefit [38]. The data in these dry AMD models highlight the involvement of both the RPE and photoreceptors and suggest that expression of the ophNdi1 gene in both RPE and photoreceptors may be preferable to that in RPE alone.
Whether a higher dose of AAV2/8-VMD2-ophNdi1 would have provided similar benefit to CMV-driven ophNdi1 in the NaIO 3 -induced mice was not investigated in the current study, as the dose used with the VMD2-driven therapy was already 6-fold higher than the highest dose of CMV-driven ophNdi1 used previously. Note that there is an increasing focus in the field of virally-delivered gene therapy on lowering dose requirements of AAV gene therapies, thereby reducing the risk of immune responses.
It is notable that an allied experimental approach has been explored previously for ABCA4-linked Stargardt disease (STGD1) in genetically modified mice. In the study, the ABCA4 gene was expressed in the RPE, but not photoreceptors, providing partial rescue of the disease and suggesting a role for both the RPE and photoreceptors in the pathogenesis of STGD1 [68]. Both that study and our own highlight the value of differential promoter constructs to explore the relative contributions of different cell types to the pathogenesis of disease and the optimal target cell population in therapeutic interventions.
In summary, while AAV2/8-VMD2-ophNdi1, which only expresses in RPE, did provide some benefit in two murine models of dry AMD, AAV2/8-CMV-ophNdi1, which expresses in RPE and rods and cones, amongst other cell types, provided more robust and consistent benefit using a variety of functional and histological assays. It remains unclear which cells are affected first in dry AMD, RPE or photoreceptor cells. However, ubiquitous and RPE-specific promoter-driven gene therapies can be used to interrogate the contribution of different cell types to disease pathogenesis and the optimal target cell population for a therapy. In the current study, the data from two murine models suggests that using a general promoter to drive expression of ophNdi1 and boosting mitochondrial function in both RPE and photoreceptor cells is more beneficial than targeting the RPE alone.

Study Design
An optimised complex I equivalent gene, ophNdi1 driven from an RPE-specific promoter, VMD2, was delivered via recombinant AAV2/8 to models of dry AMD: the Cfh −/− mouse, NaIO 3 -induced mouse and primary pRPE cells insulted with NaIO 3 or A2E/blue light. Functional benefit was determined using physiologic readouts, ERG and OKR. Histological analysis and cellular assays included mitochondrial function, ROS and morphological readouts.

Subretinal Injections, Electroretinography and Ros Assay
All animal work was performed in accordance with the European Union (Protection of Animals used for Scientific Purposes) Regulations 2012 (S.I. no. 543 of 2012) and the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals, and approved by the animal research ethics committee in Trinity College Dublin (Ref. no. 140514/240320). C57BL/6J, Cfh −/− on a pure C57BL/6J background and 129 S2/SvHsd mice (Harlan Laboratories, Blackthorn, UK.) were maintained under specific pathogenfree conditions. Injections were performed on two-month-old mice as described, except that anaesthesia comprised of ketamine and medetomidine (57 mg/kg and 0.5 mg body weight, respectively) and, following injection, an anaesthetic-reversing agent (Atipamezole Hydrochloride, 1.33 mg/kg body weight) were delivered by intraperitoneal injection [71]. An amount of 4.5 × 10 8 vg of AAV2/8-VMD2-ophNdi1 was injected into Cfh −/− mice, while contralateral eyes received the same volume (3 µL) of PBS. At 8 months, ERG responses from treated eyes were compared to fellow eyes (n = 13 mice; paired t-tests). Mice were analysed histologically at 8 months of age as described (n = 6) [38]. At 7 months post-injection, mice were sacrificed, retinal cells were dissociated and a CellRox TM Green Reagent (Invitrogen, Waltham, MA, USA) ROS assay was performed using a flow cytometry assay, as described [38]. Median levels of CellRox TM , representing relative ROS levels, were recorded. Paired t-tests of the means were performed to compare medians of treated versus untreated eyes of Cfh −/− mice (n = 10 mice).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism (version 9.4, GraphPad Software, Boston, MA, USA). t-tests and ANOVA with post-hoc Tukey were considered significant at p < 0.05.